In vivo stable isotope labeling of fruit flies reveals post-transcriptional regulation in the maternal-to-zygotic transition

Abstract

An important hallmark in embryonic development is characterized by the maternal-to-zygotic transition (MZT) where zygotic transcription is activated by a maternally controlled environment. Post-transcriptional and translational regulation is critical for this transition and has been investigated in considerable detail at the gene level. We used a proteomics approach using metabolic labeling of Drosophila to quantitatively assess changes in protein expression levels before and after the MZT. By combining stable isotope labeling of fruit flies in vivo with high accuracy quantitative mass spectrometry we could quantify 2,232 proteins of which about half changed in abundance during this process. We show that approximately 500 proteins increased in abundance, providing direct evidence of the identity of proteins as a product of embryonic translation. The group of down-regulated proteins is dominated by maternal factors involved in translational control of maternal and zygotic transcripts. Surprisingly a direct comparison of transcript and protein levels showed that the mRNA levels of down-regulated proteins remained relatively constant, indicating a translational control mechanism specifically targeting these proteins. In addition, we found evidence for post-translational processing of cysteine proteinase-1 (Cathepsin L), which became activated during the MZT as evidenced by the loss of its N-terminal propeptide. Poly(A)-binding protein was shown to be processed at its C-terminal tail, thereby losing one of its protein-interacting domains. Altogether this quantitative proteomics study provides a dynamic profile of known and novel proteins of maternal as well as embryonic origin. This provides insight into the production, stability, and modification of individual proteins, whereas discrepancies between transcriptional profiles and protein dynamics indicate novel control mechanisms in genome activation during early fly development.

Fig. 1.

Experimental strategy used to analyze the MZT. A, labeled and unlabeled yeast was used to grow embryos that were harvested before (right) and after (left) the MZT. Embryos were combined, lysed, digested, and subjected to SCX fractionation (B). Each of the 28 SCX fractions were analyzed by reversed-phase LC-MS (C), and the resulting MS and MS/MS spectra were used to, respectively, quantify and identify peptides and stored in a PostGreSQL database (D).

Fig. 2.

Expression profiles of all quantified proteins. A, scatter plot representation of protein data from the two biological independent experiments. Linear regression analysis shows good correlation (r² = 0.831) between the data sets. B, log₂ ratios of all 1,737 quantified proteins in the two experiments. Up-regulated and down-regulated proteins indicate zygotic and maternal expression, respectively, and are shown in the insets.

Fig. 3.

Comparison of data sets annotating zygotic products. A Venn diagram shows the overlap between our data set and published data to define zygotic transcripts and proteins. De Renzis et al. (6) investigated chromosome-ablated mutants to discriminate between transcriptional and post-transcriptional regulation of gene expression; Lécuyer et al. (25) used high resolution fluorescent in situ hybridization to assess differential localization of maternal and zygotic transcripts. The gene symbols indicating eight and four genes in the De Renzis et al. (6) and Lécuyer et al. (25) data, respectively, were claimed to be zygotic, but proteins were found to be down-regulated in our work, suggesting maternal expression.

Fig. 4.

Correlation of protein and transcript expression. Shown is a scatter plot representing changes in expression levels of proteins and transcripts during the MZT. The encircled region contains data points where transcript levels show decreased abundance, although protein levels do not change significantly. Of these 41 proteins, 37 have catalytic activity and are listed in additional supplemental Table 9.

Fig. 5.

Post-translational processing of PABP. Identified peptides of PABP along with spectra indicating their abundance before and after MZT are shown. Peptides originating from the N-terminal part of the protein (shown in red) show no change, apparent from equal intensities of the unlabeled and labeled peptides. This is in contrast to peptides originating from the C-terminal part of the protein (blue). The intensity of the unlabeled peptides is dramatically less compared with that of the labeled peptides, indicating truncation of this part of the protein during MZT. The four RNA recognition motifs (N-terminal part) are highlighted in gray, and the polyadenylate motif (C-terminal part) is highlighted in orange.

Fig. 6.

Protein expression validation by Western blot analysis. About 15 μg of proteins from lysates of early and late embryos (before and after MZT, respectively) as well as adult flies were subjected to SDS-PAGE and blotted. Blots were probed with three different rabbit polyclonal antibodies against PABP (A) or probed with anti-Rack1 and antieIF3- S9 antibodies (B). Subsequently blots were stripped and probed with an anti-α-tubulin antibody as a loading control.